Abstract: The continuous mutation of the SARS-CoV-2 during infection and persistent evolution has led to a decrease in the effectiveness of existing vaccines, and therefore there is an urgent need for effective vaccines capable of establishing broad-spectrum immune protection in humans. The authors used mRNA vaccine technology to design a mRNA vaccine (P0) targeting the spike protein of the Prototype strain. The Prototype-mut mRNA vaccines P1−P7 were prepared by introducing amino acid mutations into the S protein gene sequence and immunized in mice. The results showed that the S protein mRNA vaccines with Q498R-Y505H-H655Y and Q498R-N501Y mutation sites significantly increased the neutralizing antibody (NAb) titer against the Delta strain after the second vaccination (P < 0.05). The geometric mean titer (GMT) of the vaccines introducing the Q493R-Q498R-H655Y and Q498R-N501Y mutations against multiple strains of Prototype, Alpha, Beta, Delta, and Omicron BA.1 were 1∶194.0, 1∶73.5, 1∶32.0, 1∶194.0, 1. 16.0 and 1∶294.1, 1∶294.1, 1∶64.0, 1∶256.0, 1∶8.0, respectively. Meanwhile, the vaccines introducing the mutations Q493R-Q498R-H655Y, Q498R-Y505H, and Q498R-Y505H-H655Y could achieve 100% neutralizing antibody positive seroconversion rate against the four strains Prototype, Alpha, Beta, and Delta. Through the strategy of introducing point mutations in S protein mRNA vaccine, the effect of the introduction of amino acid mutation sites that may have the potential for immune escape or alteration of receptor binding affinity on the cross neutralizing activity of mRNA vaccines was verified, which provides a reference for the development of mRNA vaccines to enhance their broad-spectrum properties.